PROJECT SUMMARY/ABSTRACT Non-coding small RNAs (sRNAs), which act swiftly and specifically, are ideal gene regulators in pathogens such as Coxiella burnetii that shift rapidly between disparate environments (e.g. aerosol vs. intracellular). However, very little is known about sRNAs that facilitate the pathogenicity of C. burnetii?a select agent that causes acute Q fever and chronic endocarditis. It is important that we gain better molecular insights into C. burnetii infection because it is found across the globe and has the potential to cause epidemics such as the recent outbreak in the Netherlands that involved more than 4,000 human cases and resulted in the culling of over 50,000 goats (a primary reservoir). Our long-term goal is to define the functions of non- coding RNAs during C. burnetii infection and to apply this knowledge to developing novel therapeutic ap- proaches. Towards attaining this goal, the objective of this application is to identify sRNAs that promote C. burnetii's growth within human cells. Based on preliminary data that identified several novel sRNAs in Cox- iella, our central hypothesis is that sRNAs promote intracellular growth of C. burnetii. The objectives of this project will be accomplished by two Specific Aims: (1) Identify sRNAs that are important to Coxiella's intra- cellular growth. Using RNA-seq we expect to identify sRNAs that are expressed during various stages of in- fection, and by examining transposon-insertion mutants we will assay their importance to intracellular growth. (2) Define the role of the sRNA CbsR14 in promoting Coxiella's intracellular growth. To begin to understand how sRNAs facilitate infection, we will interrogate the function of CbsR14, This sRNA was cho- sen because its expression was induced intracellularly, a transposon insertion in it significantly reduced Coxiella's intracellular growth, and preliminary analyses indicated that it targets the adjacent yidC gene. All studies to date on C. burnetii infection have only investigated proteins; hence, the research proposed here is innovative because it will depart from the status quo to investigate the role of non-coding RNAs in facilitat- ing Coxiella infection. This study is significant because it will, for the first time, identify sRNAs that pro- mote intracellular growth of C. burnetii. This new knowledge is expected to advance the field considerably by transforming our current understanding of Coxiella's pathogenicity. Additionally, uncovering the regula- tory circuit of YidC could be applicable to other organisms because this membrane protein is conserved across bacteria and mitochondria. Furthermore, the approaches we develop in this project will guide us (and others) to analyze other classes of non-coding RNAs (e.g. riboswitches) in Coxiella, and to examine sRNAs in other intractable intracellular pathogens such as Chlamydia, Rickettsia, Anaplasma and Ehrlichia. Moreover, a deeper understanding of sRNA-based gene regulation could contribute to the development of novel anti-Coxiella therapeutic agents that target critical components of the sRNA regulon.